Study 1

Method of sterilization
-------------------------------------------------------------------------------------------------------------

1
 Study 2
Selection and collection of virological specimens
-------------------------------------------------------------------------------------------------------------
Laboratory diagnosis of viral infections requires an understanding of the pathogenesis of the
suspected agent, stage of infection, as well as age and immunocompetence of the infected
individual. It is important to select the appropriate specimens, collect the specimen carefully to
optimize recovery of the infectious agent, and transport the specimens as directed so as to
maintain viability and minimize overgrowth with contaminating organisms.

Selection of specimens
The specimen should be collected from the target organ most closely associated with clinical
symptoms to identify the etiologic agent responsible for the patient's disease.

Materials
I. Reagents
Viral transport medium (VTM) prevents specimen drying, maintains viral viability and
retards the growth of microbial contaminants. The VTM contains gelatin and
antimicrobial agents in a buffered salt solution. Tubes containing 2-3 ml VTM are used
for swab specimens, while those with 5-7 ml VTM are suitable for tissue samples.
II. Supplies
o Sterile, leak-proof, screw-cap containers including urine cups, disposable
centrifuge tubes (15 and 50 ml), and smaller tubes (e.g., 4 ml vials, 13x100
tubes), suitable for holding 1-2 ml of VTM.
o Sterile cotton, Dacron, or rayon-tipped swabs with plastic or aluminum shafts
small tip flexible (fine-shafted aluminum) swabs are used for certain samples like
urethral swabs. Do not use calcium alginate swabs or wooden-shafted swabs.
o Tuberculin syringe with 26 or 27 gauge needle for aspirating vesicular fluid
o Blood collection tubes containing anticoagulant (ACD).
III. Equipment
Refrigerator (2-8oC)

Handling instructions
In general, all specimens (except feces) must be collected in a designated sterile container and
kept cool on ice or refrigerated (not frozen). Detailed specimen collection and handling
instructions are described later.

2
Table 1: Handling instruction of the specimen with collection schedule and volume

Specimen collection guidelines for virus culture

A. Collect specimens as soon as possible after the onset of symptoms. The chance of
patient recovery is best during the first 3 days after onset and is greatly reduced beyond
5 days with many viruses. Autopsy samples need to be collected as soon as possible
after death before tissues start decomposing.
B. Refer to the specimen collection guide table for viral culture. In general, place swabs
into a tube containing a small volume of VTM, and scrapings and small pieces of tissue
into a tube containing a small volume of VTM or saline. Place fluid and bulk specimens
(e.g., tissue) into a sterile leak-proof container. Add a volume of VTM sufficient to
prevent drying of tissue.
C. Collect specimens as aseptically as possible to avoid introduction of contaminating
organisms that can take over the culture later and make accurate diagnosis difficult.
D. Place each specimen into a separate container labeled with the patient's name and
identification number, the collection site, the date of collection, and the time of the
collection.
E. Obtain a complete patient history, including the date of onset of symptoms, clinical
findings, recent exposure history, animal or arthropod contacts or bites, recent travel to
areas of endemic infections, and recent vaccines.

3
Specimen collection by type
All specimens should be collected immediately upon onset of clinical symptoms or death of the
animal.
 Feces: Place 2-4 grams inside a sterile sealed container
 Rectal swab: Insert swab 4-6 cm and roll against mucosa. Place swab in 1-2 ml of sterile
saline or viral transport media.
 Vesicle or lesion swab: Open lesion carefully using a sterile instrument. Moisten a
sterile swab with sterile saline or other transport media and collect cells from open
lesion. Place swab in 1-2 ml of sterile saline or viral transport media.
 Ocular swab: Collect from lower conjunctiva using a swab moistened with sterile
saline. Place swab in 1-2 ml of sterile saline or viral transport media.
 Corneal or conjunctiva scraping: Place scraping in 1-2 ml of sterile saline or viral
transport media.
 Nasal swab: Swab nostrils separately and place swabs in 1-2 ml of sterile saline or viral
transport media.
 *Nasopharyngeal swab: Insert sterile swab through nostril into nasopharynx and rotate
several times. Remove and place swab in 1-2 ml of sterile saline or viral transport
media.
 Oropharyngeal/Throat swab: Swab posterior throat and tonsil area and place swab in
1-2 ml of sterile saline or viral transport media.
 *Nasal Aspirate: Insert suction device through nostrils into nasopharynx. Aspirate fluid
while removing suction device. Flush device with sterile saline and collect in a sealed
container.
 Serum: Collect in red top tube. Centrifuge and remove from clot if possible.
 Whole blood: Collect in EDTA (purple top) tube.
 CSF: Collect in sterile container.
 Tissue: Place in sterile container or plastic bag with a small amount of sterile saline to
keep moist.
 Semen: Collect in semen straw and transfer immediately into liquid nitrogen.

(*Note: For antibody titers, send paired samples for the most accurate results.)

4
 Study 3
Preservation and transportation of virological specimens
-------------------------------------------------------------------------------------------------------------
Storage and shipment of clinical specimens
All specimens should be stored in sterile containers and refrigerated immediately upon
collection. Ship with enough ice packs is sufficient to maintain an approximate 4 oC environment.
NEVER send dry swabs.
Specimen transportation guidelines

A. Place the tightly-capped specimen container and associated lab requisition form into
separate compartments of a plastic specimen transport bag.
B. Deliver all specimens to the laboratory as soon as possible after collection, since loss of
infectivity occurs over time; samples containing low titers of labile viruses are most
likely to show loss of infectivity with delayed transport. If immediate delivery is not
possible, refrigerate specimens, place them on wet ice or use a cold pack to keep them
cool. Loss of viability is slower at low temperatures (2-8°C). Never froze the sample.
C. Transport specimens for respiratory syncytial virus culture immediately for prompt
inoculation into cell culture.
D. Do not freeze specimens before testing them. Cytomegalovirus and some respiratory
viruses are readily inactivated by even one freeze-thaw cycle. If samples must be frozen,
quick-freeze rapidly at -70°C or lower, not at -20°C. Store at 4°C or -70°C. Survival of
labile viruses may promote using viral transport mediums like M4 HBSS with gelatin.
Virology submission guidelines
To ensure accurate diagnosis of viral disease, it is imperative to collect the correct
specimen. The specimen should reflect the system(s) involved in clinical disease and should be
collected during the acute phase of infection when viral concentration is at its maximum. Refer
to the following information when selecting appropriate specimens for testing.
Age, sex, breed, previous history, clinical signs, any special features related to host or
environment.
Preservatives
1. Using 50% buffered glycerol / 10% dimethyl dulfoxide (DMSO)
2. Keeping frozen condition (-200C to -900C)
3. Balanced salt solution: The solution containing inorganic salts such as Na, K, Ca, Mg, Cl,
SO4, PO4, and CO3 and 1% glucose solution but it is very costly
4. Nutrient broth or tryptose broth: generally most commonly used because for economy
5. PBS (Phosphate buffer saline)
6. Normal saline or sterile distilled water.

5
General properties of virus
Viruses are obligate intracellular parasites and lack the enzyme necessary for protein and
nucleic acid synthesis. Outside of the host cells, viruses are inactive. However, inside living cells,
viruses show some of the characteristics of living things. The extracellular infectious virus
particle is called “virion”. The largest virus is pox virus (300 nm) and the smallest is parvovirus
(20 nm).The size of the virus can be measured directly with the aid of electron microscopy. The
virion consists of a nucleic acid surrounded by a protein coat, the capsid. Capsid introduces the
viral genome into host cells by adsorbing to cell surfaces of the host. A capsid is made up of
protein subunits called capsomers. Capsomers determine antigenicity of the virion. The capsid
with the enclosed nucleic acid is called as nucleocapsid. The nucleocapsid protects the nucleic
acid from the action of any chemical and environmental agents. The virion may be enveloped or
naked (nonenveloped).The envelope is the outer covering of the virus; lipoproteinaceous in
nature and is derived from the host cell membrane during the release of the progeny virus by
budding. The envelope may be covered by spikes, which are made up of carbohydrate and
protein complexes and project out into space from the surfaces of the envelope used for
attachment to host cells. Envelopes provide chemical, antigenic and biological properties on
virus (Fig 1).

Figure 1: Structure of Virus

The shape of the virus particles varies in different groups of viruses is shown below (Fig 2).

Figure 2: Rabies virus: Bullet shaped, Ebola virus: Filamentous shaped, Pox virus: Brick shaped

6
Inoculum preparation from solid and liquid sample Study 4
-------------------------------------------------------------------------------------------------------------
Preparation of viral inoculum from solid sample

From liquid sample:

7
 Study 5
Cultivation of virus
-------------------------------------------------------------------------------------------------------------
Viruses are obligate intracellular parasites so they depend on host for their survival. They
cannot be grown in non-living culture media or on agar plates alone, they must require living
cells to support their replication. The primary purpose of virus cultivation is:
o To isolate and identify viruses in clinical samples.
o Demonstration of virus in appropriate clinical specimens by culture establishes
diagnosis of viral diseases.
o To do research on viral structure, replication, genetics and effects on host cell.
o To prepare viruses for vaccine production.
o Isolation of virus is always considered as a gold standard for establishing viral
etiology of a disease.
o To increase number of viruses for different study.
o To observe manifestation produced by viruses in the living host for diagnostic
purpose.

The following systems are used for the cultivation of viruses-

1. Intact host system/Experimental animal, like guinea pig,
2. Avian embryo/Embryonated eggs and
3. Tissue culture system

Diluent
The substances which is used for diluting of the viral suspension
1. Balanced salt solution: The solution containing inorganic salts such as Na, K, Ca, Mg, Cl,
SO4, PO4, and CO3 and 1% glucose solution but it is very costly
2. Nutrient broth or tryptose broth: generally most commonly used because for economy
3. PBS (Phosphate buffer saline)
4. Normal saline
5. Sterile distilled water.

8
 Study 5A
Cultivation of the virus in intact host
-------------------------------------------------------------------------------------------------------------
Inoculation of virus in animals

Laboratory animals are widely used for routine cultivation of virus; they play an essential role in
studies of viral pathogenesis. Live animals such as monkeys, mice, rabbits, guinea pigs, ferrets
are widely used for cultivating virus. Monkeys were used for the isolation of Poliovirus. But due
to their risk to handlers, monkeys find only limited applications in Virology. Mice are the most
widely employed animals in virology. The different routes of inoculation in mice are
intracerebral, subcutaneous, intraperitoneal or intranasal. After the animal is inoculated with
the virus suspension, the animal is observed for signs of disease, visible lesions or is killed so
that infected tissues can be examined for virus.

Advantages:
1. Animal inoculation may be used as diagnostic procedure for identifying and isolating a virus
2. Mice provide a reliable model for studying viral replication
3. Give unique insight into viral pathogenesis and host virus relation
4. Used for the study of immune responses, epidemiology and oncogenesis
5. Pathological manifestation of diseases
6. Production of antisera.

Disadvantages:
1. Expensive and difficulties in maintenance of animals
2. Difficult in choosing of animals for particular virus
3. Some human viruses cannot be grown in animals or can be grown but do not cause disease
4. Mice do not provide models for vaccine development
5. It will lead to generation of escape mutants
6. Issues related to animal welfare systems
7. Limitation of the space, cages and equipment
8. Chance of cross infection among animals
9. Latent infection may misinterpret the result.

9
 Table 2: Particular intact host for cultivation of the virus

No. Animals Agents

1. Mice CNS disease, influenza virus, vesicular disease of domestic animals
2. Guineapig CNS disease, vesicular disease
3. Rabbit Pox, rabies, pseudorabies
4. Monkey Yellow fever virus, polio myelitis virus
5. Hamster CNS disease virus
6. Chicken All susceptible viruses

After inoculation of virus the laboratory animal are kept in well ventilated room with requisite
ration. Controlled animal are also to be kept separately with the similar condition to compare
the result of virus inoculation. Every day all the animals will be observed for the development of
sign and symptom. Animal which have developed the symptoms is subjected to post mortem
examination for the collection of virological samples. Samples should be collected aseptically
and kept for further studies.

Figure 3: Inoculation of virus in mice

10
Cultivation of the virus in embryonated eggs Study 5B
-------------------------------------------------------------------------------------------------------------
Prior to the advent of cell culture, animal viruses could be propagated only on whole animals or
embryonated chicken eggs. Good pasture in 1931 first used the embryonated hen’s egg for the
cultivation of virus. The process of cultivation of viruses in embryonated eggs depends on the
type of egg which is used. The egg used for cultivation must be sterile and the shell should be
intact and healthy. A hole is drilled in the shell of the embryonated egg, and a viral suspension
or suspected virus containing tissue is injected into the fluid of the egg. Viral growth and
multiplication in the egg embryo is indicated by the death of the embryo, by embryo cell
damage, or by the formation of typical pocks or lesions on the egg membranes. An
embryonated egg offers various sites for the cultivation of viruses (Fig. 4). The different sites of
viral inoculation in embryonated eggs are:
1. Chorioallantoic membrane (CAM)
2. Amniotic cavity/sac
3. Allantoic cavity/sac
4. Yolk sac
Chorioallantoic Membrane (CAM) is mainly employed in the growth of poxvirus. Virus growth
and replication in the CAM is indicated by visible lesions (pocks); grey white area in transparent
CAM. Herpes simplex virus is also grown. Each pock is derived from a single virion. The
morphology of the pocks may vary depending on the nature of the virus. Under optimal
conditions, each infectious virus particle can form one pock. Hence this method is suitable for
plaque studies. Herpes simplex virus can also be inoculated via CAM.
Allantoic Cavity is the most popular and simple method for viral inoculation. Allantoic
inoculation is employed for the growth and replication of the influenza virus for vaccine
production. This will provide a rich yield of influenza and some Paramyxo viruses. Other
allantoic vaccines include Yellow fever and rabies vaccines. Duck eggs provide a better yield of
rabies virus and were used for the preparation of the inactivated non-neural rabies vaccines.
But they need a longer incubation period than embryonated hen’s egg. Most of avian viruses
can be isolated using this method.
Amniotic Cavity: The amniotic sac is employed inoculated for primary isolation of influenza a
virus and the mumps virus. Growth and replication of virus in egg embryo can be detected by
hemagglutination assay.
Yolk Sac: It is also a simplest method for growth and multiplication of virus. Mostly mammalian
viruses are isolated using this method. Immune interference mechanism can be detected in
most of avian viruses. This method is also used for the cultivation of some bacteria like
Chlamydiae and Rickettsiae.

11
 Figure 4: Routes of virus inoculation in embryonated egg

Advantages:
a.
b.
c.
d.
e.

Disadvantages:
a.
b.
c.
d.
e.

Factors influencing the growth of viruses in avian embryo

1. Age of the embryo
2. Route of inoculation
3. Dilution of viruses
4. Volume of the inoculum used
5. Temperature of incubation
6. Time of incubation
7. Physiological and nutritional status of the embryo
8. Immune status of the flock from which the eggs were obtained
12
 Table 3: Routes and amount of inoculation for virus cultivation in avian embryo
Routes Amount Day of embryo Viruses may be cultivated
Allantoic cavity 0.1-0.2 ml 9-12 NDV, IB, Fowl Plague
CAM 0.1-0.3 ml 10-11 ILT, Fowl Pox, Duck Plague
Yolk sac 0.1-0.5 ml 6-8 Avian encephalomyelitis,
MYCOPLASMA, RICKETTSIA
Amniotic cavity 0.1-0.2 ml 10-11 Influenza
Intracerebral 0.01-0.02 ml 8-14 Rabies, pseudo rabies

After inoculation of viruses in avian embryos the embryos are to be kept in the incubator for a
period of 1-6 days. During which the embryos are to be examined daily for the development of
any changes produced by virus in the embryo. Death of embryo within 24 hours of inoculation
be treated as non specific causes (Bacterial contamination, hemorrhage, traumatic injury).
During the observation period the embryos are to be examined twice daily to record the
changes produced by the virus. After the death of the embryo, these are to be kept in the
refrigerator for few hours thereafter viruses are collected. During virus collection changes
produced by the virus are also noted.

Changes produced by virus in the embryo

1. Death
2. Hemorrhage and congestion of the subcutaneous tissue and epidermis
3. Curling and dwarfing of the embryo
4. Thickening and fibrosis of amnionic membrane
5. Pock lesions of the Chorioallantoic membrane
6. Improper development of feathers of the embryo
7. Inclusion bodies in the tissues of the embryo
8. Deposits of urates in the stroma of the kidney embryo
9. Necrotic foci in the liver and heart

Source of eggs
Fertile eggs less than 1 week old should be obtained from healthy, specific pathogen free (SPF)
breeder flocks. Eggs are usually incubated 6-11 days before being inoculated; and the length of
the incubation is determined by the route of inoculation to be used. Eggs should be candled
after 5-6 days of incubation to determine which are fertile and which are unfertile or not viable.

13
Techniques of viral inoculation in avian embryo
Allantoic sac inoculation
a) Candling (9-12 days) whether the embryo is developed or not.
b) Encircle air cell and making X mark 1/4 inch below the base of the air cell, this area is
free from blood vessel.
c) The site of inoculation disinfected with a solution of 70% ethyl alcohol.
d) A small hole is drilled through the egg shell in the marked area.
e) Using a tuberculin syringe, 0.1-0.3 ml of inoculum per egg by inserting the 27 gauge
needle vertically through the hole
f) Injecting the desired amount by pushing the entire length of the needle.
g) Following inoculation, the hole is sealed with melted paraffin or nail polish.
h) Incubate at 370C for 6 days.
Yolk sac inoculation
a) Candling (6-8 days) whether the embryo is developed or not. Encircle air cell.
b) Drilling a small hole through the egg shell along the center axis at the top of the egg.
c) Using a tuberculin syringe, 0.1-0.5 ml of inoculum per egg by inserting the 22 gauge
needle vertically through to its full length and injecting the desired amount.
d) Following inoculation, the hole is sealed with melted paraffin or nail polish.
e) Incubate at 370C for 10 days.
Chorioallantoic membrane inoculation (CAM)
a) Candling (10-11 days) whether the embryo is developed or not.
b) Encircle air cell and mark the side of the egg approximately midway along the long axis
where the vein structre is not well developed.
c) Drilling a small hole through the shell and eggshell membrane at the center of the air
cell. Drilling a second hole on the side of the egg, being careful not to perforate the egg
shell membrane in the marked area.
d) While holding the egg against the egg candler a piece of suction bulb applied to hole of
the air cell. Withdrawing the air from the air cell will cause the CAM to drop, thus
forming a new false air cell directly over the CAM.
e) Using a syringe fitted with a 25 gauge needle in tuberculin syringe, 0.1-0.3 ml of
inoculum per egg by inserting the needle vertically just inside the egg shell and injecting
the desired amount.
f) Following inoculation, the hole is sealed with melted paraffin or nail polish.
g) Incubate at 370C for 6 days.

14
 Study 5C
Cultivation of the virus in cell/tissue culture
-------------------------------------------------------------------------------------------------------------
Cell culture

The idea of cell cultures dates back to the end of the nineteenth century. It was not a practical
laboratory technique until the development of antibiotics. Cell cultures have replaced
embryonated eggs as the preferred type of growth medium for many viruses. Cell culture
consists of cells grown in culture media in the laboratory. These cultures can be propagated and
handled like bacterial cultures; they are more convenient to work with than whole animals or
embryonated eggs.

Major problem with cell cultures:

1. The process requires trained technicians with experience in working on a full time basis.
2. State health laboratories and hospital laboratories do not isolate and identify viruses in
clinical work.
3. Tissue or serum for analysis is sent to central laboratories to identify virus.

Figure 5: Cultivation of the virus in the cell line

15
Preparation of the cell/tissue culture
-------------------------------------------------------------------------------------------------------------
It is a system where tissues are maintained in a laboratory condition with the supplementation
of requisite nutrients. It is 3 types.
1. Primary cell culture
2. Continuous cell line
3. Organ culture

1. Primary cell culture: A culture started from cells, tissues or organ taken directly from an
animal until it is subcultures for the first time termed primary cell culture.

2. Continuous cell line: Tissues which are derived from transformed neoplastic tissues can be
propagated indefinitely in culture are called continuous cell culture or cell line. e.g. Vero cell
line, Baby hamster kidney cell line (BHK21 cell line), Hela cell line, Pig kidney (PK) cell culture.

3. Organ culture: Some respiratory viruses are difficult to isolate. Human rhino viruses have
been isolated on embryonic tracheal explants while tracheal ring cultures are examined for
degeneration of the epithelium & cessation of ciliary beating as indication of viral replication.

Advantages
a.
b.
c.
d.

Disadvantages
a.
b.
c.
d.

Changes in cell culture

a.
b.
c.
d.

Maintenance media
---------
Growth promoting media
-----------------
16
 Study 6
Preparation of chick embryo fibroblast cell cultures
-------------------------------------------------------------------------------------------------------------
Procedure

1) Chicken embryo with the age of 10 days old will be collected. The shell surface should
be cleaned by immersing in alcohol for 5 minutes or by swabbing with tincture of iodine.
2) Only the embryo will be collected with the removal of head and leg in a petridish
3) The embryos will be chopped finely with the help of sterile scissor and forceps.
4) The minced tissues will be transferred into an Erlenmeyer flask or side arm flask.
5) Washing the embryos 3-5 time with PBS.
6) Trypsinized the tissue for breaking the intercellular bridges (PBS + Trypsin 0.05%).
7) Incubate the tissue in incubator for 20 minutes for action of trypsin.
8) Centrifuge at 350 rpm for 10 minutes.
9) Supernatant will be discarded.
10) One ml cold calf serum will be added for 10 ml suspension.
11) Centrifugation at 1200 rpm for 5 minutes.
12) Discarding the supernatant.
13) Resuspending the cell in 10 ml medium.
14) Counting the cell.
15) Adjusting the cell yield about 2 X 106 cells per ml by adding growth medium.
16) Seeded the cells in cell culture flask and place in an incubator.

Types of dilution
A. Simple dilution: One dilution differs from other not in a constant ratio. like 1:2, 1:3, 1:5,
1:7, 1:8

B. Serial dilution: One dilution differs from other a constant ratio. Say 1:2, 1:4, 1:8, 1:16. In
calculating the potency of the virus serial dilution is calculated.
1. Two fold dilution: Equal amount of materials and diluents.
2. Five fold dilution: One part material plus 4 parts diluents.
3. Ten fold dilution: One part material plus 9 parts diluents.

17
 Study 7
Plaque assay for virus detection
-------------------------------------------------------------------------------------------------------------
One of the most important procedures in virology is measuring the virus titer (concentration of
viruses in a sample). A widely used approach for determining the quantity of infectious virus is
the plaque assay. This technique was first developed to calculate the titers of bacteriophage
stocks. Renato Dulbecco modified this procedure in 1952 for use in animal virology, and it has
since been used for reliable determination of the titers of many different viruses.

Figure 6: Formation of the plaque by virus

To perform a plaque assay, 10 fold dilutions of a virus stock are prepared, and 0.1 ml aliquots
are inoculated onto susceptible cell monolayers. After an incubation period, to allow virus to
attach to cells, the monolayers are covered with a nutrient medium containing a substance,
usually agar that causes the formation of a gel. When the plates are incubated, the original
infected cells release viral progeny. The spread of the new viruses is restricted to neighboring
cells by the gel. Consequently, each infectious particle produces a circular zone of infected cells
called a plaque. Eventually the plaque becomes large enough to be visible to the naked eye.
Dyes that stain living cells are often used to enhance the contrast between the living cells and
the plaques. Only viruses that cause visible damage of cells can be assayed in this way. An
example of plaques formed by poliovirus on a monolayer of HeLa cells is shown at figure 6. In
this image, the cells have been stained with crystal violet, and the plaques are readily visible
where the cells have been destroyed by viral infection.

The titer of a virus stock can be calculated in plaque forming units (PFU) per milliliter. To
determine the virus titer, the plaques are counted. To minimize error, only plates containing
between 10 and 100 plaques are counted, depending on the size of the cell culture plate that is
used. Statistical principles dictate that when 100 plaques are counted, the sample titer will vary
by plus or minus 10%. Each dilution is plated in duplicate to enhance accuracy.
In the example shown below, there are 17 plaques on the plate made from the 10 -6 dilution.
The titer of the virus stock is therefore 1.7 x 108 PFU/ml.

18
Staining of the virus
Study 8
-------------------------------------------------------------------------------------------------------------
Materials required:
- EM grid-grade tweezers (2 or more pairs)
- Uranyl acetate
- Ultra-purified water (e.g. Milli-Q water)
- Petridish
- 4x 2 ml microcentrifuge tubes with screw caps
- 4 ml plastic culture tube with cap
- Small stir bar
- Stir plate
- Filter paper (cut into wedges)
- 0.02 µm syringe filter and syringe (3-5 ml)
- Lab coat, respiratory protection (mask), eye protection
- Timer
- Waste container for uranyl acetate

Positive staining of viruses on grids: This is the easiest staining to do. It is “quick and dirty”. It
provides less detail of your viruses, but will allow you to measure their dimensions. It is not the
proper staining method if you want really pretty micrographs of your viruses.

Negative staining of viruses on grids: Negative staining is equal parts magic, art, luck, and skill.
If done correctly, it will yield detailed views of your viruses and if you are very lucky, it will yield
a few pretty micrographs of your viruses.

Negative staining is one of the simplest, rapid and economical methods for preparing certain
biological specimens, in this case a suspension of viral particles, for transmission electron
microscopy. This method relies on a heavy metal "stain" (usually phosphotungstic acid) coating
the virus particle outlining its surface structure. Stain may also penetrate cavities within the
virus revealing internal structure. Variation in size and details of surface and internal
architecture are used for identification, a technique that becomes a useful and rapid diagnostic
method in a number of clinical situations. A few examples of negatively stained viruses are
provided to illustrate the ultrastructural features and diagnostic utility of this technique.

19
Negative staining of viruses
-------------------------------------------------------------------------------------------------------------
Negative Staining of Viruses on Grids

 Make 2 ml of 2% uranyl acetate with ultra-purified water in a 4 ml culture tube using a

stir bar to mix the sample on a stir plate for 30 minutes to 1 hour. DO NOT TURN ON
THE HEAT.
 Filter the uranyl acetate solution through a 0.02 µm syringe filter into a 2 ml screw cap
tube. This removes any particles of uranyl acetate that have not fully dissolved.
 Hold the grid in EM grid grade tweezers.
 Using a pipettor, drip 3 drops of uranyl acetate solution onto the shiny side of the grid,
allowing each of them to run off into the uranyl acetate waste container.
 Add a third drop of uranyl acetate solution to the shiny side of the grid and allow it to sit
for 45 seconds (Fig. 7).
 Wet a wedge of filter paper in ultra purified water. It should be wet, not soaking wet.
 Using the wedge of wet filter paper, wick away the stain from the grid but leave a thin
sheen of stain on the grid. This is the magic/skill part too much stain left on the grid and
all you will see in the TEM is stain too little stain left on the grid and your viruses will not
be negatively stained.
 Set the tweezers with the grid on a petridish such that the grid does not touch anything
(Fig. 8)
 Wait until the grid is dry (~1 hr). This step is optional. The grid can dry in the grid box if
you are in a hurry.
 Place the grid in the grid box and store the grid box in a desiccator.

Figure 7. Staining the grid with a drop of uranyl acetate. Figure 8. Drying the grid.

WARNING: if you put the grid in the desiccator before it has dried, the stain will look “cracked”

20
Purification of virus
Study 9

-------------------------------------------------------------------------------------------------------------

21
Inactivation of viruses Study 10
-------------------------------------------------------------------------------------------------------------

22
Preparation of 1% erythrocyte (chicken RBC) suspension
-------------------------------------------------------------------------------------------------------------
Procedure
1. An anticoagulant such as 4% sodium citrate (one part to four parts blood) or Alsever's
solution (equal volumes) should be in the syringe into which the blood is drawn.
2. After it is mixed gently, the blood is transferred slowly to a large, conical centrifuge tube
for washing.
3. An equal amount of phosphate-buffered saline (PBS) is added and the suspension is
centrifuged at 1500 rpm for 5 minutes.
4. The supernatant is poured off, and 20-30 volumes of PBS are added to the packed cells.
5. The cells are resuspended gently and the centrifugation step repeated two times more.
6. The cells can then be used to prepare the 1% suspension based on volume by adding 1
ml of the packed cells to 100 ml of PBS.
7. The cells should be kept in chiller and can be used for a week.

Hemagglutination is a naturally occurring activity with avian influenza virus Newcastle disease
virus, adeno virus, and infectious bronchitis virus.

Equipments and materials

 1% red blood cell suspension
 PBS
 Pipettes
 Multichannel pipettor and tips (to measure 25 L)
 96- well plastic microtitre plates with V bottom wells.

Hemagglutination test procedure

Generally two fold dilution is used

1. Using the multichannel pipettor, dispense 25 l diluent of PBS into each well of two
(sample in duplicate) rows of the microtitre plate.
2. Place 25 L of virus suspension (for example, allantoic fluid) into the first well of each
row (This is a 1:2 dilution).
3. Mix well and make two fold dilutions of the suspension across the row by transferring
25 L of fluid from one well to the next. Discard 25 L from the last well of each row so
that the volumes in each well will be the same.
4. Dispense 25 L PBS into each well of a control row. This row will show the normal
settling patterns and time of red blood cells in suspension.

23
 5. Add 25 L PBS to each well (including the control wells).
6. Add 25 L of 1% red blood cell suspension to each well.
7. Mix gently and allow standing at room temperature for 45 minutes. Cover the plate to
stop dehydration.
8. The plates are to be shaken well.
9. These are to be incubated at room temperature for 45 minutes.
10. After that the test is to be observed.
11. Buttoning in the bottom of the hole indicates no hemagglutination and no buttoning
indicates hemagglutination.
12. End point is to be considered 1 HA unit.

FOR EXAMPLE if the sixth well (1:64) is the last to show hemagglutination, then the original
material contained 64 (26) HA units.

Definition of 1 HA unit
One HA unit is defined as the highest dilution of antigen which will completely agglutinate a
test dose of red blood cells under standard conditions of temperature and time of incubation.

HA titre
The reciprocal of the end point of the virus suspension that cause agglutination of the specific
amount of blood cell.

24
The anatomy of the embryonated egg
-------------------------------------------------------------------------------------------------------------
Shell and shell membrane: The membrane is closely attached to the shell. Together they
function as an exchange system and gaseous and liquid molecules pass in both directions. This
is why eggs must be incubated in humid conditions. Eggs incubated in low humidity will lose
moisture and eventually the embryo will die.
Air sac: Eggs have a rounded and a pointed end. The air sac is the space at the rounded end and
has a function in respiration and pressure adjustments.
Chorioallantoic membrane and allantoic cavity: The membrane is attached to the embryo and
functions to remove soluble, insoluble and gaseous waste products. As the embryo develops,
the sac increases in size. The sac contains allantoic fluid into which Newcastle disease virus is
shed after inoculation of the allantoic cavity. The fluid is harvested for vaccine production.
Some viruses are propagated by inoculation of the Chorioallantoic membrane. This involves
placing inoculum on the membrane.
Yolk sac: This is also attached to the embryo and contains the nutrient-rich yolk. As the embryo
develops, the yolk sac decreases in size to approximately 1 cm diameter 3 days before the
embryo hatches.
Amniotic Sac: This sac surrounds the embryo. It is filled with liquid and serves to protect the
embryo against physical damage as well as functioning as an area of exchange of molecules. As
the embryo develops, the membrane stretches and the amniotic sac is barely visible in the fully
developed embryo.
Albumen: The egg white, which consists mainly of protein.

To examine the structure of embryonated eggs at various stages of development, remove the
entire contents of the egg into a petridish.

Figure 9: The anatomy of a ten day old embryonated egg

25
Candling of the eggs
-------------------------------------------------------------------------------------------------------------
Introduction
Candling is the process of holding a strong light above or below the egg to observe the embryo.
A candling lamp consists of a strong electric bulb covered by a plastic or aluminium container
that has a handle and an aperture. The egg is placed against this aperture and illuminated by
the light. If you do not have a candling lamp, improvise. Try using a torch.
Candling is done in a darkened room or in an area shielded by curtains.
Determining the viability of the embryo
Under the candling lamp, the embryo appears as a dark shadow with the head as a dark spot.
Healthy embryos will respond to the light by moving. Sometimes the movement is very sluggish
and it can take 30 to 40 seconds for the embryo to move when held under the candling lamp.
This indicates the embryo is not healthy and the egg should be discarded.
Look carefully at the blood vessels. They are well defined in a healthy embryo. After an embryo
has died, the blood vessels start to break down. Eggs with cracked shells should be discarded.
Infertile eggs: These are easy to detect, as the egg is clear. Discard
Early deaths: The embryo has developed for several days and then died. Candling will reveal a
small dark area and disrupted blood vessels. Often deteriorating blood vessels will appear as a
dark ring around the egg. Discard.
Late Deaths: It is often difficult to tell apart from a viable embryo at the same stage of
development. Look for the absence of movement and breakdown of the blood vessels. Discard.
Viable Embryos: These move in response to the light and have well defined blood vessels. Mark
the air sac and the inoculation site and then return the eggs to incubator ready for inoculation.

Marking the inoculation site:

1. Hold the blunt end of the egg against the aperture of the candling lamp and note the position
of the head of the embryo.
2. Turn the egg a quarter turn away from the head.
3. Draw a line on the shell marking the edge of the air sac.
4. Draw an X approximately 2 mm above this line.
5. The X marks the inoculation site.

Note: In some eggs the air sac will have not developed on the blunt end but half way down the
egg. These eggs are not suitable for vaccine production. They can be used for inoculation during
routine titrations to establish infectivity titres.

26
 Infertile egg Early death of embryo: Late death of embryo:
Embryo is small and does Blood vessels may have
not move. Blood vessels started to break down.
have broken down. Embryo does not move.

Viable embryo:
Strong healthy blood vessels. Marking the inoculation site. Candling an egg.
Embryo moves.

Figure 10: Features of embryonated eggs visible during candling

27
Syllabus for Virology (MIPA-258)

Course Content Comments

Selection and collection of viral samples.
Transportation and preservation of viral sample for laboratory diagnosis.
Inoculums preparation for virological study.
Preparation of inoculum from solid and liquid sample.
Purification of virus by physical methods.
Purify virus by chemical methods.
Purify virus by immunological methods.
Negative staining for morphology study of virus.
Cultivation of virus in intact host system, embryonated eggs, and cell
culture.
Preparation of cell/tissue cultures.
Description, requirements and procedure of plaque assay.
Inactivation of viruses.
Group representative (GR): Course Tutor (s):

28
29